Human immunodeficiency virus (HIV) was identified as etiological agent of AIDS independently in Paris and in Washington (Barre-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzinoux C., Rozenbaum W., Montagnier, L. (1983) Science 220, 868-871; Popovic, M., Sarngadharan, M. G., Read, E., Gallo, R. C. (1984) Science 224, 497-500). In last 18 years, the efforts towards the development of active virostatics lead to the inventions of many medicaments, that strongly contributed to the treatment of this disease. Despite that, the scientists still did not succeed in controlling and stopping the spread of the disease. AIDS is still a serious worldwide problem. Especially in developing countries and in central Africa the epidemy has catastrophical extent endangering the basic social and national principles.
Human immunodeficiency virus (HIV) belongs to the genus Lentivirus, family Retroviridae. Viruses belonging to this family contain diploid RNA genome and use reverse transcriptase for their replication. Retroviruses are further divided into three genera: oncoviruses, lentiviruses and spumaviruses (Gelderblom, H. R., P. A. Marx, M. Ozel, D. Gheysen, R. J. Munn, K. I. Joy, and G. Pauli (1990) Morphogenesis and Fine Structure of Lentiviruses. In Pearl, Lawrence (Ed.). Retroviral Proteinases: Control of Maturation and Morphogenesis). The genus Lentivirus involves viruses causing slow chronical diseases. The most important representatives are HIV-1, HIV-2 (both further referred to under common abbreviation HIV) and the simian lentivirus SIV.
Mature virion HIV is a spherical particle with the diameter of 100 to 110 nm. The nucleus of the virus, encapsulated by capsid protein, consists of two copies of the genomic single-stranded RNA, nucleocapsidic proteins (NC) and viral enzymes reverse transcriptase (RT), integrase (IN) and protease (PR). The external capsid consists of phospholipid membrane derived from the host cell. Button-shaped structures, consisting of three molecules of glycosylated surface protein SU loosely embedded into transmembrane protein TM, extrude from the capsid (Gelderblom, H. R., P. A. Marx, M. Ozel, D. Gheysen, R. J. Munn, K. I. Joy, and G. Pauli (1990) Morphogenesis and Fine Structure of Lentiviruses. In Pearl, Lawrence (Ed.). Retroviral Proteinases: Control of Maturation and Morphogenesis).
HIV genome is formed by two identical RNA molecules of the size of about 9.2 kb, encoding for 9 various genes. The basic structure of the genome, characteristic for all the retroviruses, consists of three structural genes gag, pol and env. In addition to structural genes, 6 genes encoding for proteins with regulatory functions, participating in the virus' replication, were identified in the HIV genome.
Replication cycle of the HIV inside the host cell can be divided into several stages (Carrasco L., Sonenberg N., Wimmwe E. (1993) Regulation of Gene Expression in Animal Viruses, ed. by L. Carrasco, et al., Plenum Press, New York): surface glycoprotein of the viral capsid SU recognizes and binds with high affinity the protein receptor CD4+, which is expressed on the surface of T-lymphocytes. For effective binding a coreceptor, specific according to the host cell type, is necessary. Virus enters the cell by endocytosis or by fusion of the virus capsid with the cell surface and the content of the capsid gets into the cell cytoplasm. The reverse transcriptase (RT) transcripts the viral RNA into the double-stranded DNA, that is integrated into the host cell chromosome by the enzyme integrase (IN). So the virus persists in the idle state (latent infection) until the moment of activation and transcription of viral genes by the host RNA polymerase II. According to the proviral mRNA, viral polyprotein precursors Gag and Gag-Pol are synthesized at the ribosomes. Posttranslationally modified polyproteins and genomic RNA are collected close to the cell surface and during the process called budding the virions are released from the cell. In the immature particle, the polyprotein precursors Gag and Gag-Pol are cleaved by virus-encoded protease (PR), yielding functional proteins, creating thereby the mature infectious particle. If the HIV PR is afflicted or its activity is inhibited, the virion remains immature.
Most extensively examined is HIV-1 PR. It is a dimeric aspartate protease, consisting of two identical non-covalently bonded subunits. The primary structure of the monomeric subunit consists of 99 amino acids. The most important contribution to the knowledge of the HIV-1 PR secondary structure were the crystallographic structural analyses (Wlodawer, A., Miller, M., Jaskólski, M., Sathyanarayana, B. K., Baldwin, E., Weber, I. T., Selk, L. M., Clawson, L., Schneider, J., Kent, S. B. (1989) Science 245, 616-621), that disclosed the double rotational C2 symmetry and high content of β-structures. Four of the chains in the core of the molecule form a leaf of the shape of the “letter Ψ”, that is characteristic for all the aspartate proteases. Triplet of the active site (Asp25-Thr26-Gly27) is placed in the bend of the protein chain and its structure is stabilized by hydrogen bonds net.
HIV PR must first be autocatalytically cleaved out of the polyprotein precursor and subsequently it cleaves the precursor in nine exactly defined sites. HIV PR specifically cleaves the viral polyprotein despite the fact, that the amino acid sequences of the sites cleaved rather differ. In contrast to other endopeptidases (pepsin, trypsin, renin), that hydrolyze peptide bonds next to particular amino acids, in the HIV PR no analogous relation to the primary structure can be defined. Instead, specificity stemming from the cumulative effect of independent mostly weak interactions among individual side chains of the substrate and the corresponding subsite of the enzyme is assumed. Important is the effect of hydrophobic interaction, surface, polarity, potential of the secondary structure etc. (Poorman, R. A., Tomasselli, A. G., Heinrikson, R. L., Kézdy, F. J. (1991) J. Biol. Chem. 266, 14554-14561).
HIV Protease Inhibitors
Several steps of the HIV life cycle were chosen as the targets of therapeutical treatment. The most important are the reverse transcriptase (dideoxynucleosides, their analogs and non-nucleoside inhibitors), binding and entry of the virion into the host cell (soluble CD4 receptors and their derivatives, polyanionic compounds, fusion inhibitors), integration of the provirus by the integrase into the host chromosome, regulation of the transcription by protein products of the genes tat and rev etc. (review: De Clercq, E. (1998) Collect. Czech. Chem. Commun. 63, 449-479). Maturation of the retrovirus and above all its most important enzyme HIV protease, that is the object of this patent application, is also the object of extensive research and the rational designing of medicaments. In the Czech Republic seven inhibitors were or are going to be approved for clinical use: saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir and atazanavir. All these inhibitors competitively inhibit binding of the natural substrates to HIV PR and decrease the infectivity of the virus by blocking the virion maturation.
Development of the Resistance Against the HIV PR Inhibitors
Launching the protease inhibitors in the years 1995-1996 and introduction of HAART—highly active antiretroviral therapy lead to moderation of onset of opportunic infections and decrease in mortality. This had increased the hope of patients and physicians for developing sufficient therapy of AIDS. Unfortunately, soon after the launch of the new medicaments also their limits were found.
When PR inhibitor is applied, it suppresses the virus replication. If the replication is not fully suppressed, a small population of the virus, which is resistant to the inhibitor, can survive under the selection pressure. This leads to the resistance of the virus (Larder, B., Richman, D., Vella, S. (1998) HIV resistance and implications for therapy, MediCom Inc., Atlanta, USA). Until now, the mutations in at least 49 positions in 99-amino acid monomer were observed (Gulnik, S., Erickson, J. W., Xie, D. (2000) Vitam. Horm. 58, 213-256).
The essential factors responsible for quick development of resistant varieties are natural variability of HIV genome (polymorphism) and dynamic virus replication during the latent phase and during the idle state (Erickson, J. W., Burt, S. K. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 545-571). The genetical variability of HIV probably stems from the combination of high error rate of reverse transcriptase, genome recombination and selection pressure of human immunity system.
There are several possible strategies, that virus can use in order to develop its resistance against protease inhibitors. Among the most important are: mutations in the binding site of the enzyme, which directly influence binding; mutations outside the binding site of the enzyme, which indirectly influence binding; mutations of the cleaved sites of the HIV PR in polyprotein substrates. Also mutations that decrease the stability of the HIV PR dimer and thereby its affinity to the inhibitor can contribute to the resistance, and finally also mutations outside the protease area that can result for example in more effective shift of the reading frame (Erickson, J. W., Burt, S. K. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 545-571; Boden, D., Markowitz, M. (1998) Antimicrob. Agents Chemother. 42, 2775-2783).
The HIV protease inhibitors known so far can be divided into three basic groups: (i) compounds designed as isosters of the substrate transition state (statine, hydroxyethylaminic, hydroxymethylenic, hydroxyethylenic type, α,α′-difluoroketones, etc.) (ii) compounds proposed with the aid of rational design on the basis of geometric similarity to the substrate (e.g. DMP inhibitors) (iii) compounds having accidental structural similarity to the substrate, obtained by the screening of natural substances isolated e.g. from biological material (Lebon F., Ledecq M. (2000) Curr. Med. Chem. 7, 455-477).
Design of viral protease inhibitors is a non-trivial problem. In contrast to many other enzymes, whose substrate is a simple organic molecule (e.g. the natural substrate of nitrogen(II) oxide synthase is L-arginin, so simple modifications like Nω—OH—Arg {hacek over (c)}i Nω-Me-Arg are succesful inhibitors), the design of HIV protease inhibitors is much more complicated issue. Its natural substrate is polypeptide, that is recognized in a specific site and cleaved, yielding functional enzymes and structural proteins of the virion. Theoretically, both by methods of molecular modelling and by ab initio calculations, and also practically the design of specific HIV protease inhibitor represents difficult problem, when low-molecular substrate must show higher affinity to the enzyme than natural polypeptide does.
All HIV protease inhibitors used in the treatment (saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir and atazanavir) can be classified into group (i). They have peptidomimetic character and are competitive inhibitors of the active site of the enzyme. Such compounds usually show unfavourable pharmacodynamic properties (in order to reach the effective concentration level in infected cells it is necessary to administer orally rather high doses of the medicaments) and later the resistance can develop. In order to overcome the resistance problem there is considerable effort devoted to discovering non-peptide inhibitors, that could be developed by rational design on the basis of structural information, obtained by rentgenostructural analysis of complexes of HIV PR and known inhibitors, eventually by screening of combinatorial or other libraries of chemical compounds.